CN106831956A - A kind of antineoplastic polypeptide MUDP 21 and its application - Google Patents

Abstract

The invention provides it is a kind of can killing tumor cell polypeptide and its application, its amino acid sequence such as SEQ ID NO:Shown in 1；A kind of antineoplastic polypeptide and its application are further related to, the antineoplastic polypeptide includes tumor cytotoxicity domain and wears spanning domain, the amino acid sequence such as SEQ ID NO of tumor cytotoxicity domain:Shown in 2.Antineoplastic polypeptide of the invention wears spanning domain in itself without cytotoxicity, but after connection tumor cytotoxicity domain, has and obvious suppresses tumor proliferation, the effect of migration invasion and attack.Antineoplastic polypeptide of the invention, can not only be expected to suppress tumour with reference to other treatment mode separately as antineoplastic biotherapeutics.

Description

A kind of antineoplastic polypeptide MUDP-21 and its application

Technical field

The present invention relates to neoplasm targeted therapy field, more specifically it relates to it is a kind of can killing tumor cell polypeptide and its Using.

Background technology

In recent years, the morbidity and mortality of malignant tumour rise year by year, it has also become developed country and partial development China Family's population main causes of death, are one of global maximum common problems, seriously threaten human health and life security.Cause This, the common concern and great attention treated by international community of cancer, and find small the resisting of safe and effective, adverse reaction and swell Tumor medicine is always the focus of biomedical research.Traditional clinical drug therapy is by direct killing tumour cell and then suppression The growth of tumour, but generally there is toxicity to normal cell, produce the side effects, Er Qiesui such as bone marrow suppression, immunologic hypofunction The extension of medicine use time, tumour cell is also easy to produce the resistance to the action of a drug.Therefore, from different approaches find effect it is good, toxicity is low, spy The strong antineoplastic of the opposite sex is the main target of current oncotherapy.

Biologically active peptide (bioactivity peptides), is one also known as Functional Polypeptides (functional peptides) Class has the peptide of physiological active functionses, with suppression growth of tumour cell or the weight such as antibacterial, antiviral, hypotensive, hypoglycemic Act on.Because its molecular weight is small, be easy to absorption, the exploitation of health products and medicine is can be widely applied to.

Accordingly, it would be desirable to build one kind can targets neoplastic cells, the novel polypeptide of born of the same parents can be efficiently entered again.

The content of the invention

To solve problem above, inventor is prepared for a kind of biologically active peptide, and the polypeptide and cell-penetrating peptide are passed through Covalent bond is coupled together, and reaches existing targets neoplastic cells, and with efficiently entering the effect of born of the same parents.

Based on the research, the invention provides it is a kind of can killing tumor cell polypeptide, its amino acid sequence such as SEQ ID NO:Shown in 1.

Present invention also offers it is above-mentioned can killing tumor cell application of the polypeptide in antineoplastic is prepared.

Present invention also offers a kind of antineoplastic polypeptide, it includes tumor cytotoxicity domain and wears spanning domain, institute State the amino acid sequence such as SEQ ID NO of tumor cytotoxicity domain:Shown in 1.

Preferably, the amino acid sequence for wearing spanning domain such as SEQ ID NO:Shown in 2.

Preferably, it is described to wear the N-terminal that spanning domain is connected to the tumor cytotoxicity domain

Present invention also offers application of the above-mentioned antineoplastic polypeptide in antineoplastic is prepared.

It is an advantage of the current invention that the spanning domain of wearing of antineoplastic polypeptide of the invention does not have cytotoxicity in itself, but even After connecing tumor-killing domain, there is the obvious effect for suppressing tumor proliferation, migrating invasion and attack.Antineoplastic polypeptide of the invention, no Can only be expected to suppress tumour with reference to other treatment mode separately as antineoplastic biotherapeutics.

Brief description of the drawings

Fig. 1 is to be incubated the fluorescent microscopy images after tumour cell with MUDP-21 and control polypeptide；

The statistical chart that Fig. 2 influences for the MUDP-21 of various concentrations on SH-SY5Y cell-proliferation activities；

Fig. 3 is the culture after the fluid nutrient medium culture SH-SY5Y cells two weeks respectively containing MUDP-21 and control polypeptide The violet staining photo in hole；

Fig. 4 is the statistical chart of the number of cell clones calculated according to Fig. 3；

After Fig. 5 is soft agar medium culture SH-SY5Y cells 3-4 weeks respectively containing MUDP-21 and control polypeptide The Methyl thiazoly tetrazolium assay stained photographs of culture hole；

Fig. 6 is the statistical chart of the number of cell clones calculated according to Fig. 5

Fig. 7 is cell scratch experiment detection MUDP-21 and control polypeptide cell migration capacity；

Fig. 8 is the violet staining photo of Transwell experiments；

Fig. 9 is the cell count figure of the Transwell experiments of the IMR32 cells counted to get according to Fig. 8.

Specific embodiment

Principle of the invention and feature are described below in conjunction with example, example is served only for explaining the present invention, and It is non-for limiting the scope of the present invention.

1. the synthesis of antineoplastic polypeptide

Synthesize a kind of antineoplastic polypeptide by solid-phase synthesis, it includes a tumor cytotoxicity domain and one is worn Spanning domain, wherein tumor cytotoxicity domain sequence such as SEQ ID NO:Shown in 1, spanning domain sequence such as SEQ ID are worn NO:Shown in 2, the N-terminal of tumor cytotoxicity domain is connected to, resulting sequence is：Amino acid sequence is YGRKKRRQRRR-METRWGTDGVLMTAVIGAGSC(SEQ ID NO:3), it is named as MUDP-21.In order to study conveniently, I Also antineoplastic polypeptide C-terminal mark marked by fluorescein isothiocyanate FITC.

The cellular localization detection of 2.MUDP-21

Human neuroblastoma cells' strain SH-SY5Y cells are respectively adopted to be tested.The cell kind that logarithmic phase is grown It is implanted on the cover glass in 24 orifice plates, per about 8000-12000, hole cell.With 10% hyclone, the training of DMEM in high glucose culture medium Support, and add 60 μM of polypeptide, cultivate 24 hours.With the incorrect order after the amino acid path reorganization with the polypeptide same composition Peptide is used as control peptide.Using polypeptide of the laser confocal microscope detection with fluorophor positioning in the cell, take pictures simultaneously Preserve.Experimental result can effectively penetrate tumour cell as shown in figure 1, MUDP-21 is adding cell culture system 24 hours afterwards Film enters cell, and strong endochylema and karyon dyeing is presented, and illustrates that the polypeptide can be efficiently entering in the endochylema and karyon of tumour cell.

3.MTT colorimetrically analysings detect inhibitory action of the MUDP-21 to SH-SY5Y cell growths

Take the logarithm the SH-SY5Y cells in growth period, after being digested with 0.25% pancreatin, add corresponding complete medium end Only digestion and re-suspended cell, count and adjust the concentration of cell suspension to 5 × 10⁴Individual/ml, is subsequently adding in 96 orifice plates, per hole 100μl.Culture plate is placed in 37 DEG C of constant temperature, 5%CO₂Cell culture incubator in cultivate.

After culture 24 hours, cell is completely adherent, suction out waste and old nutrient solution, and it is that 200 μ l contain difference to add final volume The DMEM basal mediums of concentration MUDP-21, with out of order polypeptide as control group, 37 DEG C of constant temperature, 5%CO are placed in by culture plate₂'s Cultivated in cell culture incubator.

Nutrient solution is suctioned out after 48 hours, is washed 2 times with PBS, add the μ l of Methyl thiazoly tetrazolium assay (MTT) solution 20 of 5mg/ml With the μ l of fresh basal medium 180；In 37 DEG C of constant temperature, 5%CO₂Cell culture incubator in cultivate；After 4 hours, discard and contain MTT Nutrient solution, add 150 μ l DMSO after on microoscillator vibrate 15 minutes.

The light absorption value OD in each hole is measured at 490nm used in enzyme-linked immunosorbent assay instrument₄₉₀, and calculate inhibiting rate：Cancer cell is given birth to Inhibiting rate (%) long=[(control group OD- blank group OD)-(administration group OD- blank group OD)]/(control group OD- blank group OD) × 100。

Experimental result as shown in Fig. 2 MUDP-21 can significantly inhibit the growth activity of SH-SY5Y cells, and in dose-dependant Property, IC₅₀It is 60 μM, and control peptide unrestraint is acted on.

4. the inhibitory action that colony formation detection MUDP-21 breeds to SH-SY5Y cells

Take the logarithm the SH-SY5Y cells in growth period, digested with 0.25% pancreatin and blow and beat into individual cells.Cytometer Number, and adjust cell concentration with culture medium.Cell suspension is made into gradient multiple dilutions, it is thin with 100,200,500, every hole respectively The Graded Density of born of the same parents is inoculated in six orifice plates containing 2ml culture mediums, and is shaken gently for, and cell is uniformly dispersed.MUDP-21 is pressed According in 60 μM of concentration addition culture mediums.Culture plate is placed in 37 DEG C of constant temperature, 5%CO₂Cell culture incubator in cultivate 2 weeks or so.

When occurring macroscopic clone in culture hole, terminate culture.Nutrient solution is discarded, is washed 3 times with PBS.Add 4% Paraformaldehyde room temperature fixes cell 15 minutes, then discards paraformaldehyde, is washed 3 times, every time 5 minutes with PBS.Add appropriate crystallization Purple, dyeing sucks dye liquor after 30 minutes, then washes away dyeing liquor with clear water, is air-dried.

As shown in Figures 3 and 4,60 μM of MUDP-21 can significantly inhibit the proliferation activity of SH-SY5Y cells to experimental result.

5. soft-agar cloning forms the inhibitory action that experiment detection MUDP-21 breeds to SH-SY5Y cells

Take the logarithm the SH-SY5Y cells in growth period, digested with 0.25% pancreatin and gently blown and beaten, make unicellular, Make viable count, 300 cells are added per hole.Prepare 1.2% and 0.7% two molten point fine jade of concentration respectively with distilled water Lipolysaccharide liquid, after autoclaving, maintains 40 DEG C of temperature, with Anti-solidification.

By MUDP-21 according in 60 μM of concentration addition culture mediums.By 1:1 ratio trains 1.2% agarose and 2 × DMEM After supporting base (containing 2 × antibiotic and 20% calf serum) mixing, take in 3ml mixed liquors six orifice plates of addition, cooled and solidified is made Bottom-layer agar.By culture plate be placed in 37 DEG C of constant temperature, 5%CO2 cell culture incubator it is standby.

By 1:After 1 ratio makes 0.7% agarose and the mixing of 2 × DMEM culture mediums, appropriate cell suspension is added, filled Divide and mix, injection is covered with the culture plate of bottom-layer agar, gradually form double agar layers.After after top-layer agar solidification, constant temperature is placed in 37 DEG C, 5%CO₂Cell culture incubator in cultivate 3-4 weeks.

To 5mg/ml MTT solution is added in six orifice plates, after 4 DEG C are dyeed 4 hours, observation photograph is taken out.Count cell clone Form number (each at least 50 cell of clone).

Experimental result is as it can be seen in figures 5 and 6,60 μM of MUDP-21 can significantly inhibit the proliferation activity of SH-SY5Y cells.

6. scratch experiment detects inhibitory action of the MUDP-21 to SH-SY5Y cell migrations

Draw horizontal line to be marked at the six orifice plate back sides with marking pen；After cell dissociation, take appropriate cell and be inoculated with, will Culture plate is placed in 37 DEG C of constant temperature, 5%CO₂Cell culture incubator in cultivate.

After cell covers with plate bottom, compare ruler with 100 μ l pipette tips, perpendicular to the horizontal line cut of culture plate behind；Suck Cell culture fluid, is washed 3 times with PBS, washes away the cell of suspension；Add serum free medium, Taking Pictures recording.Culture plate is placed in perseverance Temperature 37 DEG C, 5%CO₂Cell culture incubator in after culture 24 hours, Taking Pictures recording again.

Experimental result is as shown in fig. 7,60 μM of MUDP-21 can significantly inhibit the migratory activity of SH-SY5Y cells.

Inhibitory action of the 7.Transwell experiments detection MUDP-21 to SH-SY5Y cell migrations

Added in 24 well culture plates and contain complete medium, Transwell cells are put into hole；Take the logarithm growth period SH-SY5Y cells, are digested with 0.25% pancreatin and gently blown and beaten, and make unicellular, make viable count.Added per hole 2.5×10⁵Individual cell, and 60 μM of polypeptide is added to upper chamber；24 orifice plates are placed in 37 DEG C of constant temperature, 5%CO₂In cell culture incubator, Culture 24 hours.

Transwell cells are taken out, nutrient solution in hole is discarded, is washed 2 times with PBS；Methyl alcohol fixes 20 minutes, by cell wind It is dry；0.1% violet staining 30 minutes, the cell that upper strata does not pass through aperture is dabbed off with cotton swab；Random five under microscope Visual field observation of cell, numeration.

Experimental result is as shown in FIG. 8 and 9：60 μM of polypeptides can significantly inhibit the migratory activity of SH-SY5Y cells.

The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all it is of the invention spirit and Within principle, any modification, equivalent substitution and improvements made etc. should be included within the scope of the present invention.

Sequence table

<110>Wuhan Union Hospital

<120>A kind of antineoplastic polypeptide MUDP-21 and its application

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<170> PatentIn version 3.5

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<213>Artificial sequence

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Met Glu Thr Arg Trp Gly Thr Asp Gly Val Leu Met Thr Ala Val Ile

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Gly Ala Gly Ser Cys

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<213>Artificial sequence

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Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

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<213>Artificial sequence

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Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Met Glu Thr Arg Trp

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Gly Thr Asp Gly Val Leu Met Thr Ala Val Ile Gly Ala Gly Ser Cys

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Claims (6)

1. it is a kind of can killing tumor cell polypeptide, it is characterised in that amino acid sequence such as SEQ ID NO:Shown in 1.

2. described in claim 1 can killing tumor cell application of the polypeptide in antineoplastic is prepared.

3. a kind of antineoplastic polypeptide, it is characterised in that including tumor cytotoxicity domain and wear spanning domain, the tumour is thin Born of the same parents kill the amino acid sequence such as SEQ ID NO of domain:Shown in 1.

4. antineoplastic polypeptide according to claim 3, it is characterised in that the amino acid sequence for wearing spanning domain is such as SEQ ID NO:Shown in 2.

5. the antineoplastic polypeptide according to claim 3 or 4, it is characterised in that it is described wear spanning domain be connected to it is described swollen The N-terminal of cytotoxic effect domain.

6. application of the antineoplastic polypeptide any one of claim 3-5 in antineoplastic is prepared.